Phytic acid or phytate (myo-inositol hexakisphosphate) is the primary storage form of phosphorus in most plants and is abundant in seeds and legumes. However, the monogastric animals cannot utilize phosphorous from phytate due to the lack of necessary enzymes in digestive tract. Supplementation of inorganic phosphates is a solution used to compensate the shortage in phosphorus ingestion but the excessive phosphorus in animal excretion has caused environmental pollution. In addition, the insoluble complexes formed by the highly negatively charged phytate with proteins and metal ions are major anti-nutritional factors. Phytase can hydrolyze phytate to lower inositol phosphates to release inorganic phosphate and thus has been widely applied in animal feeds to increase phosphorus availability and reduce phosphorus pollution. To date, phytase is estimated to account for 60% of feed enzyme products for increasing nutrient absorption and also reducing 50% phosphorus excretion.
Classified by protein structure and catalytic property, there are four types of phytases including histidine acid phosphatases (HAPs), protein tyrosine phosphatase (PTP)-like phytases, purple acid phosphatases (PAPs) and β-propeller phytases (BPPs), with a majority of the characterized enzymes belonging to HAP. From previous studies, the crystal structures of all families except for PAP have been solved. Among the characterized phytases, Escherichia coli phytase (EcAppA), a member of the HAP family, has drawn much attention. First, EcAppA has high specific activity (up 56 to 2000 U/mg) under the favorable pH profile for feed additive. Second, large scale production of EcAppA in an industrial strain of Pichia pastoris has been successfully achieved by using fermentor for commercial applications. However, the need to enhance the thermostability of EcAppA still remains.
Molecular engineering is a powerful approach to modify enzyme performances. Directed evolution involving random mutagenesis which builds a library provides a large pool of mutants for subsequent screening for useful mutants. But the efficiency is low and the procedure is laborious. A more ideal way is rational design, which is realized by the increasing information of protein structure and the development of powerful bioinformatics tools. Major obstacle in conducting a successful rational design is how to choose the useful residues or structural features.
According to previous studies, thermostable proteins have more hydrogen bonds and salt bridges between side chains of amino acids. In addition, based on structure and characteristic analysis of mutated T4 lysozymes, it is observed that protein thermostability can be improved by increasing stability of hydrophobic center, enhancing hydrogen bonds and salt bridges, or adding disulfide bond. More and more studies use computer simulations to improve protein thermostability, and the mutated protein structures establish a set of parameters by computer analysis. These parameters can be used for narrowing the screening scope of mutation positions when improving thermostability of other target proteins.
Therefore, the present invention intends to add disulfide bond of phytase by gene modification, so as to improve thermostability and further increase industrial value of phytase.